Summary: Chronic infection with hepatitis B virus (HBV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Globally there are an estimated 257 million persons infected with HBV. In the United States, there are 1.25 million affected individuals and the epidemiology of the infection is changing due to immigration of persons from endemic regions. The natural history of chronic hepatitis B (CHB) also appears to be changing with an increasing prevalence of HBeAg negative chronic hepatitis B and recognition of a group of patients with moderate levels of HBV DNA and ALT levels with undetermined natural history. Knowledge of the rate of disease progression among individuals with moderate levels of HBV DNA and ALT levels is unknown. Despite the availability of safe and effective oral nucleoside analogues for treatment of CHB, therapy remains problematic due to the need for prolonged therapy and limited effectiveness and tolerability of the alternate treatment, interferon. Clearance of hepatitis B surface antigen (HBsAg) is the desired surrogate endpoint of therapy but is rarely achieved with current therapy. Identifying the optimal regimen, defining when to treat, for how long and when to stop therapy to achieve this endpoint are major unresolved issues. In addition, defining the best parameters to monitor patients both on and off therapy are not clear. Hypotheses/problems addressed: 1) Define the host, viral and environmental factors that determine the natural history and outcome of HBV infection. Hepatitis B core antigen (HBcAg) and HBsAg are two viral proteins that can be stained for on liver biopsy. Their expression has been shown to correlate with viral replication but whether their expression and pattern of distribution is associated with outcome in chronic hepatitis B (CHB) or is affected by nucleos(t)ide analogue (NA) treatment is unknown. In this analysis we demonstrated that the frequency of HBcAg but not HBsAg is related to viral replication. HBcAg and HBsAg staining decrease with nucleos(t)ide analogue treatment but their patterns remain unchanged. A scattered granular pattern of HBsAg expression may be associated with development of HCC and a contiguous granular pattern of HBsAg expression may predict decreased likelihood of HBsAg loss during NA treatment. Clearance of HBsAg from serum is the most desirable endpoint and a proposed definition of functional cure. Data on long-term durability of HBsAg loss is lacking. Additionally, it is controversial whether the development of anti-HBs is required for maintenance of HBsAg loss. We assessed the durability of spontaneous or treatment-related (interferon or NA) HBsAg loss in a cohort of patients followed by the Liver Diseases Branch. We reported that HBsAg loss either spontaneous or treatment-related was durable. Anti-HBs seroconversion increased over time and appeared to be more frequent after interferon treatment. HBsAg loss is a robust endpoint for functional cure. A manuscript was accepted for publication. 2) Develop and evaluate novel, safer and more effective therapies for chronic viral hepatitis. Current therapy of CHB remains less than optimal. Relapse is common if treatment is discontinued after one year in the absence of HBsAg loss. Consequently, NAs must often be administered long-term or indefinitely. However, long-term use is associated with increased risk of side effects and higher costs. Therefore, the focus of current studies is to develop strategies to induce HBsAg loss (functional cure) to permit discontinuation of therapy and improve outcome of the infection. We are taking several approaches to this problem. The first approach is to combine peginterferon alfa with tenofovir compared to tenofovir alone. This is being conducted as part of the Hepatitis B Research Network and is a multicenter trial that enrolled 200 subjects and is ongoing. The second approach is to treat patients in the immunetolerant phase of the infection. Recent data suggest that patients in this phase of the infection may have preserved immune response perhaps due to younger age and small pilot studies suggested a high rate of HBsAg loss with a combination of interferon alfa with a nucleoside analogue. We conducted a trial to evaluate the safety and efficacy of the combination of entecavir and peginterferon in adults in the immunetolerant phase of chronic HBV infection. Unfortunately, none of the patients met the primary endpoint of both HBeAg loss and HBV DNA 1,000 IU/mL 48 weeks following cessation of therapy. Given the limited efficacy in adults in the immunetolerant phase of chronic HBV infection this approach cannot be recommended. A manuscript was accepted for publication. A third approach is to add peginterferon to ongoing long-term nucleos(t)ide analogue therapy for a period of 6 month with the premise that long-term NA therapy may partially restore the host immune response and increase efficacy of peginterferon therapy. This trial explored the mechanism of action of interferon during therapy of chronic hepatitis B. We demonstrated that administration of peginterferon was associated with induction of interferon stimulated genes and a certain genetic signature correlated with reduction in hepatitis B surface antigen levels during therapy. A fourth approach is to use relatively high doses of hepatitis B antibody in the form of hepatitis B immunoglobulin to bind HBsAg. This study will address the fundamental question of the role of HBsAg in the host immune response. We postulate that high levels of circulating HBsAg may disrupt the innate immune response and its removal may lead to partial restoration of the host immune response that is characteristic of patients with chronic hepatitis B. An approved study to administer hepatitis B immunoglobulin followed by peginterferon is planned to start enrolling in the fall. 3) Elucidate the viral pathogenesis of HBV infection and assess novel markers of HBV disease activity. The underlying pathogenesis behind the clinical phases of hepatitis B is poorly understood. Determining the presence versus absence of inflammation can be challenging. We hypothesized that circulating microRNA (miRNA) profiles would be unique between states of active inflammatory and quiescent hepatitis B and might provide valuable insight into disease pathobiology. Using Nanostring miR-array to interrogate serum samples from the hepatitis B research network with distinct clinical phenotypes we identified distinct miRNAs associated with active hepatitis compared to quiescent disease. These miRNAs hold promise as biomarkers and as clues to disease pathogenesis. Among the identified miRNAs, several were associated with clinical features and one identified miRNA, miR-194-5p, may serve as an important, non-invasive predictor of disease activity. Furthermore, miR-194-5p appears to inhibit the host anti-viral and pro-fibrotic response and is highly characteristic of the immune tolerant stage of CHB. Several new markers of hepatitis B virus activity have recently been identified. These include HBV RNA, hepatitis B core related antigen and ultrasensitive quantitative HBsAg. The Liver Diseases Branch is participating in a large multicenter study, the Hepatitis B Research Network, to define the natural history of HBeAg positive and negative chronic hepatitis B. This study has enrolled 2,000 patients at 13 North American sites. Longitudinal monitoring of this large cohort has allowed us to monitor changes in HBV phenotypes over time. We plan to assess the role of these novel HBV markers in this well characterized cohort of patients that were prospectively followed to determine their role in correctly identifying HBV phenotypes and to predict changes in phenotype over time. These assays may improve monitoring of patients with CHB.